The present invention relates to a diagnostic assay method for detecting the presence of a target nucleotide sequence (either DNA or RNA) in a biological sample, and to a polynucleotide reagent complex therefor.
Conventional methods for detecting the presence of a particular polynucleotide in a biological sample typically involve immobilization of nucleic acid of the sample on a surface as the initial step. Once the sample is immobilized, a probe polynucleotide strand, itself labeled or subsequently labeled (see EPA No. 63,879 (1982)) is incubated with the immobilized sample so as to bind to the immobilized sample by purine/pyrimidine base sequence-specific complementary base pairing when the immobilized sample contains the target nucleotide sequence. After washing off the labeled probe which has not so hybridized, the presence or absence of label on the support is then determined. Techniques for this determination include exposure of a photographic film, liquid scintillation counting, and fluorescence microscopy. With subsequent labeling (e.g., by avidin-enzyme conjugate binding to biotin on the probe) various enzyme-amplified read-outs may be used.
Other known methods involve solution hybridization of a labeled probe polynucleotide to the target nucleotide sequence of single-stranded sample polynucleotides, followed by separation and detection of labeled probe/sample polynucleotide hybrids. See PCT WO No. 84/02721 of Kohne (1984). Another assay involving solution hybridization of a labeled probe to the target nucleotide sequence is disclosed in co-pending U.S. Ser. No. 790,671 of Ellwood, et al., filed Oct. 23, 1985.
A group of inventors including M. Collins have filed a series of applications describing a displacement polynucleotide assay, reagent complexes therefor and kits for such assays including such reagent complexes. See, e.g., U.S. Ser. No. 607,885 of Diamond, et al, filed May 7, 1984, 684,305 of Collins, et al, filed Dec. 20, 1984 and U.S. Ser. No. 684,308 of Williams, et al filed Dec. 20, 1984; also see EPA No. 164,876 to be published Dec. 18, 1985, and EPA No. 167,238, to be published Jan. 8, 1986. Such assays and reagent complexes all employ:
(a) a target binding region of the probe polynucleotide which is capable of complementary base pair binding to the target nucleotide sequence to be detected, and PA1 (b) a labeled polynucleotide bound in the reagent complex to the probe, generally by complementary base pair binding to a portion of the target binding region. PA1 (a) providing a reagent complex of (i) of labeled probe polynucleotide which is capable of base pair binding via hydrogen bonds of purine/pyrimidine bases to the target nucleotide sequence, and (ii) a second polynucleotide which is bound by base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the labeled probe polynucleotide in a region of the labeled probe polynucleotide at least partially coextensive with the region in which the labeled probe polynucleotide is capable of binding to the target nucleotide sequence; PA1 (b) contacting the reagent complex with a sample under conditions in which the target nucleotide sequence, if present, binds to the labeled probe polynucleotide and displaces second polynucleotide from the labeled probe polynucleotide; PA1 (c) separating labeled probe polynucleotides from which second polynucleotide have been displaced from intact reagent complex; and PA1 (d) determining the presence and/or amount of labeled probe polynucleotides which have been separated. PA1 (i) a labeled probe polynucleotide which is capable of base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the target nucleotide sequence, and PA1 (ii) an immobilized or immobilizable second polynucleotide which is bound by base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the labeled probe polynucleotide in a region of the labeled probe polynucleotide at least partially coextensive with the region in which the labeled probe polynucleotide is capable of base pair binding to the target nucleotide sequence; PA1 (a) a polynucleotide strand having: PA1 (b) a detectable tag which is within or adjacent to the target binding region segment.
While in such system, the probe and labeled polynucleotide are generally separate molecules, certain embodiments provide that they may be segments of a single polynucleotide strand, whose preferred means of manufacture is described in U.S. Ser. No. 729,504 of Fritsch, et al. In any event, such assay methods all involve determining the displaced labeled polynucleotide or labeled polynucleotide which is not displaced.
U.S. Ser. No. 777,657 of Fritsch and Williams, filed Sept. 19, 1985, describes reagent complexes with a labeled probe polynucleotide and a second polynucleotide bound thereto in at least a portion of the target binding region. The target binding region contains a half restriction site and the second polynucleotide has at least one mismatch opposite to the half restriction site. The target nucleotide sequence displaces the second polynucleotide from the target binding region and forms an endonuclease cleavage site where the mismatch had previously been. Enzymatic cleavage of such endonuclease cleavage site (but not intact reagent complexes) is followed by separation of labeled cleavage fragments from intact reagent complex, normally by employing an immobilized or immobilizable probe.